Journal of Chromatography B, 940 (2013) 142–149

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Determination of amantadine in biological fluids using simultaneous derivatization and dispersive liquid–liquid microextraction followed by gas chromatography-flame ionization detection Mir Ali Farajzadeh a,∗ , Nina Nouri a , Ali Akbar Alizadeh Nabil b a b

Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran Toxicology Laboratory, Food Control Laboratory, Food and Drug Department, Tabriz University of Medical Science, Tabriz, Iran

a r t i c l e

i n f o

Article history: Received 20 April 2013 Accepted 26 September 2013 Available online 3 October 2013 Keywords: Dispersive liquid–liquid microextraction Derivatization Amantadine Gas chromatography

a b s t r a c t A one-step derivatization and microextraction technique for the determination of amantadine in the human plasma and urine samples is presented. An appropriate mixture of methanol (disperser solvent), 1,2-dibromoethane (extraction solvent), and butylchloroformate (derivatization agent) is rapidly injected into samples. After centrifuging, the sedimented phase is analyzed by gas chromatography-flame ionization detection (GC-FID). The kind of extraction and disperser solvents and their volumes, amount of derivatization agent and reaction/extraction time which are effective in derivatization/dispersive liquid–liquid microextraction (DLLME) procedure are optimized. Under the optimal conditions, the enrichment factor (EF) of the target analyte was obtained to be 408 and 420, and limit of detection (LOD) 4.2 and 2.7 ng mL−1 , in plasma and urine respectively. The linear range is 14–5000 and 8.7–5000 ng/mL for plasma and urine, respectively (squared correlation coefficient ≥ 0.990). The relative recoveries obtained for the spiked plasma and urine samples are between 72% and 93%. Moreover, the inter- and intra-day precisions are acceptable at all spiked concentrations (relative standard deviation 0.990. The repeatability and reproducibility of the proposed method, expressed as relative standard deviation (RSD %), were evaluated by performing the method on six repeated

Fig. 5. GC-FID chromatograms of (a) drug-free urine sample, (b) urine sample of male volunteer, (c) urine sample of female volunteer (urine samples were obtained within 24 h from the first oral administration), and (d) spiked drug-free urine sample with 300 ng mL−1 of amantadine. In all cases, the derivatization/microextraction method was performed and 0.5 ␮L of the sedimented phase was injected into GC. The analyte peak is indicated by an arrow.

samples (for intra-day) and four samples (for inter-day) at concentration of 50 ng mL−1 and were found to vary between 2.8% and 6.8%. By comparing with the direct injection of amantadine in the mixture of derivatization agent/extraction solvent, the EF of analyte in spiked de-ionized water, urine, and plasma samples were 442, 420, and 408, respectively. Excellent EnFs were obtained for amantadine by performing the proposed method (1680, 1596, and 1550 for de-ionized water, urine, and plasma samples, respectively). Furthermore, the sensitivity of the methodology was checked through the LODs and LOQs and showed that amantadine can be determined in the concentration range of ng mL−1 in the completely complex samples such as urine and plasma. Robustness of the method was evaluated at different volumes of the derivatization agent (14, 15, and 16 ␮L), and the various pHs (9.9, 10.0, and 10.1). The differences between the obtained results were less than 10%. By considering the above mentioned characteristics, it can be conclude that the presented method is sensitive, repeatable, and reproducible which is applicable in the determination of amantadine in biological samples. 3.10. Real sample analysis The utility of the proposed method was tested by analyzing plasma and urine samples of a 26-years old female and a 71-years

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Fig. 6. Total ion current (TIC) chromatograms of (a) de-ionized water spiked with 300 ng mL−1 of amantadine, (b) plasma sample of female volunteer after 12 h from drug-taking, (c) mass spectra of derivatized amantadine, and (d) mass spectra of scan 1994 (retention time 19.623 min) in plasma sample. In both cases, the derivatization/microextraction method was performed and 0.5 ␮L of the sedimented phase was injected into GC–MS.

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Table 1 Analytical features of simultaneous derivatization/DLLME followed by GC-FID determination of amantadine. Martix

De-ionized water Urine Plasma a b c d e f g h

LRa (ng mL−1 )

2.0–5000 8.7–5000 14–5000

R2 b

LODc (ng mL−1 )

LOQd (ng mL−1 )

EF ± SDe

EnF (%) ± SDf

ER (%) ± SDg

0.996 0.991 0.990

0.60 2.7 4.2

2.0 8.7 14

442 ± 21 420 ± 14 408 ± 29

1680 ± 80 1596 ± 53 1550 ± 110

62 ± 3 59 ± 2 57 ± 4

RSD (%)h Intra-day

Inter-days

2.8 4.6 5.1

3.1 6.3 6.8

Linear range. Square of correlation coefficient. Limit of detection, S/N = 3. Limit of quantification, S/N = 10. Mean enrichment factor ± standard deviation, EF = CSed /C0 , (n = 3). Mean enhancement factor ± standard deviation (n = 3). Mean extraction recovery ± standard deviation (n = 3). Relative standard deviation (n = 6, C = 50 ng mL−1 ) for intra-day and (n = 4, C = 50 ng mL−1 ) for inter-days.

old male volunteers after oral administration of amantadine (100 mg, twice a day). Figs. 4 and 5 show typical GC-FID chromatograms of these samples. It can be seen that the method is suitable for the analysis of amantadine in biological fluids and there is no interfering peak in the retention time of derivatized amantadine in the blank samples. The obtained concentrations of amantadine in plasma samples of female and male volunteers were 126 ± 11 and 110 ± 9 ng mL−1 , respectively. Also the obtained concentrations of amantadine in urine samples of female and male volunteers were 46 ± 4 and 42 ± 4 ng mL−1 , respectively. In all cases three determinations (n = 3) was performed on samples using standard addition method. The proposed procedure is so sensitive that drug content of plasma and urine was still detectable even after 12 or 24 h. Hence, this method can be used for the clinical testing and pharmacokinetic studies. In order to study the matrix effect, plasma and urine samples (both free of analyte) were diluted 10 and 5-fold, respectively, and spiked with the analyte at three concentration levels. The relative recoveries were calculated by comparing the obtained peak area of analyte in diluted samples with that of de-ionized water spiked at the same concentrations after the application of this method. The obtained relative recoveries in the plasma and urine samples were in the ranges of 72–83% and 81–93%, respectively (Table 2). The presented derivatization/microextraction procedure followed by GC–MS was performed on the plasma and urine samples to identify the observed peak in the retention time of analyte in these samples. Fig. 6 shows the typical GC–MS chromatograms of standard

Table 2 Relative recoveries obtained by simultaneous derivatization/DLLME in human plasma and urine samples spiked at different concentrations. Spiked concentration (ng mL−1 )

Relative recovery (%) ± standard deviation (n = 3) Plasma

Urine

50 100 500

72 ± 3 78 ± 2 83 ± 6

81 ± 4 90 ± 3 93 ± 3

solution of amantadine and plasma sample after performing the method on them. The mass data in Fig. 6(c) and (d) confirmed the presence of derivatized amantadine in volunteers’ plasma. 3.11. Comparison of the proposed method with others To assess the performance of method, its analytical parameters were compared with those of the other methods used in the analysis of amantadine. Table 3 summarizes LR, LOD, R2 , EF, RSD%, and extraction time of some analytical methods along with the proposed method. In comparison to the other methods for the determination of amantadine, the proposed method shows high EF, good LR, relatively low LOD, and good precision. It should be noted that in the case of 7th and 8th methods, mass spectrometry was used as detection system which is inherently more sensitive than FID. Compared with LLE, the present method consumes less sample and

Table 3 Comparison of the presented method with other methods used in the preconcentration and determination of amantadine. Sample

LRa (ng mL−1 )

R2 b

EFc

LODd (ng mL−1 )

RSDe (%)

Extraction time (min)

Method

Ref.

Human plasma Human urine Human plasma Human plasma Human urine Rat plasma Human plasma Human serum Human plasma Human urine

20–1000 5–250 2.4–201.4 2.0–60 – 50–5000 3.9–1000 0.02–5 14–5000 8.7–5000

0.990 0.990 – – 0.998 – 0.9979 – 0.990 0.991

53 54 – – – – – – 408 420

7.2 1.6 2.3 0.5 75 20 1.17 0.02 4.2 2.7

5.8 7.6 6.2 1.6 7.3 5.5 8.43 6 5.1 4.6

20 20 30 180 4 25 – – 8 8

HF-LLLME-CD-IMSf HF-LLLME-CD-IMS LLE–GC-ECDg LLE–MEKC-LIFh LLE–HPLC-UVi LLE–HPLC-UV LLE–LC–MSj LC–MS-MSk DLLME–GC-FIDl DLLME–GC-FID

[18] [18] [25] [9] [5] [26] [27] [28] This method This method

a b c d e f g h i j k l

Linear range. Square of correlation coefficient. Enrichment factor. Limit of detection. Relative standard deviation. Hollow fiber-liquid–liquid–liquid microextraction-corona discharge-ion mobility spectrometry. Liquid–liquid extraction–gas chromatography-electron capture detection. Liquid–liquid extraction–micellar electrokinetic chromatography-laser-induced fluorescence detection. Liquid–liquid extraction–high performance liquid chromatography-ultraviolet detection. Liquid–liquid extraction–liquid chromatography–mass spectrometry. Liquid chromatography–tandem mass spectrometry Dispersive liquid–liquid microextraction–gas chromatography-flame ionization detection.

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organic solvents. Another advantage of the method is the simultaneous derivatization and extraction of amantadine without heating while most of the other methods need heating during derivatization. Also, derivatization/extraction time of the presented method in comparison to other methods is very short (except 5th method) due to the large surface area of contact between the extraction solvent, derivatization agent and the sample solution. These results indicate that the simultaneous derivatization and DLLME is a fast, simple, and sensitive technique that can be used for the determination of amantadine in the human plasma and urine samples. 4. Conclusions In this study, the simultaneous derivatization/DLLME was established for the preconcentration of amantadine in biological fluids followed by GC-FID determination. The method shows relatively low matrices effects in the completely complex samples. The developed method provided several advantages including simplicity, less solvent and time-consuming, low detection limits and high EF and EnF for the determination of amantadine at ng mL−1 levels in the human urine and plasma samples. Acknowledgment The authors would like to thank the Research Council of the University of Tabriz for their financial support. References [1] R. Suckow, J. Chromatogr. B 764 (2001) 313. [2] Ch. Winek, W. Wahba, T. Balzer, Forensic Sci. Int. 122 (2001) 107.

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Determination of amantadine in biological fluids using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection.

A one-step derivatization and microextraction technique for the determination of amantadine in the human plasma and urine samples is presented. An app...
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